Multi-focal light-sheet structured illumination fluorescence microscopy system

Abstract

A multi-focal light-sheet structured illumination system that can be implemented as a part of a commercial fluorescence microscope or a module that is adaptable to fit a number of commercially available microscopes. The system provides simultaneous capture of 2D images from multiple planes within a 3D volume, which are resolved laterally and axially to provide improved resolution along the three dimensions (x,y,z). A Wollaston prism allows several axially-localized high-contrast structure illumination patterns to be generated.

What is claimed is: 
     
       1. A structured illumination system for a fluorescence microscope, comprising:
 an incoherent light source configured to produce a light beam; 
 a first linear polarizer configured to receive the light beam from the incoherent light source; 
 a plurality of equidistant parallel slits configured to be illuminated by the light beam after passing through the first polarizer; 
 a Wollaston prism comprising two birefringent wedges, each birefringent wedge with an optical axis, configured to divide the light beam into two spherical waves to generate a plurality of light-sheet structured patterns; 
 a first converging lens with a front focal plane, configured to receive the divided light beam after passing through the Wollaston prism, wherein the front focal plane is collinear with the plurality of equidistant parallel slits; and 
 a second linear polarizer configured to receive the light beam after passing through the first converging lens but prior to illuminating a sample. 
 
     
     
       2. The system of  claim 1 , wherein plurality of equidistant parallel slits are disposed in a binary mask with a vertical axis. 
     
     
       3. The system of  claim 2 , wherein the optical axes of the birefringent wedges are orthogonal to each other, and both optical axes are oriented at an angle of about 45 degrees with respect to the vertical axis of the binary mask. 
     
     
       4. The system of  claim 3 , wherein the first linear polarizer is oriented at an angle of about 45 degrees with respect to both optical axes. 
     
     
       5. The system of  claim 2 , the binary mask further comprising a horizontal axis, wherein displacement of the Wollaston prism orthogonal to the vertical axis of the binary mask and parallel to the horizontal axis of the binary mask produces a controlled shifting of the plurality of light-sheet structured patterns. 
     
     
       6. The system of  claim 2 , further wherein alteration of the orientation of the plurality of light-sheet structured patterns is achieved by joint rotation of the binary mask, the first linear polarizer, a quarter-wave plate, and the Wollaston prism. 
     
     
       7. The system of  claim 1 , wherein the incoherent light source comprises a quasi-monochromatic light-emitting diode, a white lamp with a narrow bandwidth filter, or a coherent light source with a rotating diffuser, wherein the rotating diffuser is configured to destroy a spatial coherence of the coherent light source. 
     
     
       8. The system of  claim 1 , further comprising a quarter-wave plate disposed between the Wollaston prism and the second linear polarizer. 
     
     
       9. The system of  claim 8 , further comprising a polarization rotator disposed between the quarter-wave plate and the second linear polarizer. 
     
     
       10. The system of  claim 1 , further wherein alteration of the orientation of the plurality of light-sheet structured patterns is achieved by a tunable image rotator positioned after the Wollaston prism. 
     
     
       11. The system of  claim 1 , wherein the plurality of light-sheet structured patterns further comprise a tunable spatial-period tuned by adjusting the axial separation between the plurality of equidistant parallel slits and the Wollaston prism. 
     
     
       12. The system of  claim 1 , wherein the plurality of light-sheet structured patterns comprises an axial confinement that is inversely proportional to the number of equidistant parallel slits. 
     
     
       13. The system of  claim 1 , further comprising
 a single objective lens; and 
 a recording system, wherein the recording system comprises a plurality of cameras for the simultaneous detection of multiple focal planes. 
 
     
     
       14. The system of  claim 1 , wherein the illumination system is configured to retro-fit a plurality of fluorescent microscopes. 
     
     
       15. A method for obtaining super-resolved images with high optical-sectioning capability, comprising the steps of:
 producing a light beam from an incoherent light source; 
 passing the light beam through a first linear polarizer and a plurality of equidistant parallel slits; 
 subsequently splitting the light beam by a Wollaston prism into two spherical waves with orthogonal polarization for each of the equidistant slits to generate a light-sheet structured pattern; 
 subsequently passing the beam through a converging lens and a second polarizer; 
 after passing the beam through the second polarizer, illuminating a sample with the beam; 
 capturing images from the sample through a recording system; and 
 processing the captured images. 
 
     
     
       16. The method of  claim 15 , wherein the recording system comprises a plurality of cameras. 
     
     
       17. The method of  claim 16 , further comprising the steps of:
 simultaneously illuminating a plurality of transverse sections of the sample; 
 simultaneously capturing a plurality of focal planes of the sample by the plurality of cameras. 
 
     
     
       18. The method of  claim 17 , further comprising the steps of:
 axially scanning the volume of the sample; 
 capturing a plurality of two-dimensional images from the plurality of focal planes within the three-dimensional volume of the sample; and 
 resolving the captured two dimensional images laterally and axially to create super resolved images along three dimensions. 
 
     
     
       19. The method of  claim 18 , further comprising the steps of:
 capturing three separate two-dimensional images from each of the plurality of focal planes. 
 
     
     
       20. The method of  claim 15 , further comprising the steps of:
 capturing a plurality of three-dimensional forward images; 
 decomposing the plurality of three-dimensional forward images; 
 applying a Wiener filter to the decomposed images for deconvolution and shifting; and 
 combining the filtered images to form restored super-resolved images. 
 
     
     
       21. The method of  claim 15 , further comprising the step of tuning the light-sheet structured pattern by axially displacing the Wollaston prism.